Na/Ca exchanges are powerful Ca transporters which strongly influence cardiac contractile and electrical activity, as well as secretion and contraction in other tissues. Our goal is an improved understanding of cardiac (NXC1) Na/Ca exchange function and regulation. Coupled to this goal are two further goals which transcend the scope of the proposal: the development of improved methods to study ion transport, including electroneutral transporters; and an understanding of phosphatidylinositol signaling in the control of surface membrane ion transport. 1) Transport function. To allow structure-function studies by a chimeric approach, the altered transport and gating properties of NCX1 homologues from squid (NCX-SQ1), drosophila (Calx), and arabidopsis will be analyzed. To verify an alternating access model of ion transport, we will test whether individual residues, mutated to cysteine or histidine change their accessibility from inside to outside when ions are transported. 2) Exchanger regulation. The molecular basis of exchanger modulation by phosphatidylinositol-4'-5'-biphosphate (PIP2) will be analyzed, and exchanger regulatory mechanisms will be explored in light of preliminary data indicating potential regulation at the levels of (a) type 2 phosphatidylinositol-4'-kinase, (b) Gq-regulated phospholipase C, (c) lipid phosphatases, and (d) regulatory changes of PIP2 affinity. 3) Cellular function. The hypotheses will be tested that changes of cardiac frequency and stretch modulate Na/Ca exchange activity, and thereby contractility, via changes of PIP2. Our proposed studies has three significant methodological components: 1) Giant-patch and whole-cell methods for studies of Na/Ca exchange regulatory have been improved. 2) Fiber optic methods have been developed to monitor ion transport activity via concentration changes in an artificially restricted extracellular space. They will be validated by measurements of Na/H exchange activity, and they will be used to test for possible exchange stoichiometry changes and to verify different ion selectivities of the Arabidopsis exchanger. 3). A sensitive, non-radioactive method has been developed to quantify phospholipids, including phosphatidylinositides, on TLC plates. In total, therefore, our proposal is of broad physiological significance and is highly relevant to pathological roles of Ca in cardiac ischemia.